The regulation of signaling between neurons depends on the frequency of neurotransmitter release and quantity of neurotransmitter present in the synapse. Efficient termination of signaling is required for resetting the state of the synapse for a subsequent event of neurotransmitter release. The reuptake of neurotransmitter molecules by presynaptic neurons and glial cells depends on membrane-embedded transporter proteins. These enzymes transport neurotransmitters from the extracellular space into the cell and therefore up a concentration gradient. To power this energetically unfavorable process, these transporters also move sodium ions down their concentration gradient and into the cell. Although it is well-established in humans that neurotransmitter transporters directly regulate psychological states such as anxiety and reward-seeking, much less is known about the mechanisms of substrate recognition and inhibition by clinically relevant antagonists. These efforts have been hampered by the absence of atomic-resolution structural information for any eukaryotic neurotransmitter transporters. This application details strategies to express and purify a human neurotransmitter transporter for crystallization and structural determination via x- ray diffraction. The primary goal of this application is to uncover the three-dimensional structure of a neurotransmitter transporter in complex with neurotransmitter and antagonists. This advance will provide the locations of binding sites and generate testable hypotheses to probe how transporters specifically recognize their substrates and are inhibited by antagonists. PUBLIC HEALTH RELEVANCE: Psychoactive drugs and antidepressants are thought to act on neurotransmitter transporters located in neuronal cells in the brain. These transporters normally absorb neurotransmitters back into neuronal cells, and misregulation of this activity can lead to drug addiction and psychological disorders. This application seeks to better understand how drugs affect neurotransmitter transporters by both abused and therapeutic drugs in the hope that more effective compounds can be developed.